Generation of AGM-derived Akt-EC ( AGM-EC )


 During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.


Introduction
Derivation of endothelial cell lines from embryonic or adult endothelium that supports hematopoiesis or hematopoietic stem cells (HSC) in vivo is useful for de ning niche components required for both maintenance and expansion of HSC in vitro as well as de ning what might be required to convert induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) to HSCs. It has been previously shown that AGM-derived endothelium support in vitro culture of HSCs 4 . In addition, previous studies showed that constitutive expression of AKT in endothelial cells promoted EC survival in serum-free conditions and that these AKT-expressing endothelial cell lines support the survival and expansion of adult bone marrow HSCs 5,6 . Our lab has generated and used AKT-expressing ECs from the AGM as a supporting stroma to convert AGM derived hemogenic endothelium (HE) from as early as embryonic day 9 (E9) to HSCs and to further increase HSC numbers [1][2][3] . Herein we provide a protocol for generating AGM AKT-EC to provide an in vitro vascular niche that supports the serum-free co-culture of AGM/Para-aortic splanchnopleura mesoderm derived HE. Endothelial Mitogen (Alfa Aesar, BT-203) *no longer available -currently, substituting rmVEGF-10ng/ml, rmFGF-10ng/ml, rmIGF-1-10ng/ml, and rmEGF-10ng/ml in place of endothelial mitogen) 2. Harvest embryos from pregnant females at 9.5 to 11.5 days post coitum (dpc), depending on the desired stage for obtaining EC. We typically have generated AGM-EC by this protocol from embryos at late embryonic day 10 to early day 11 (35-45 somite pairs).

Reagents
3. Dissect the AGM from the embryos in ice-cold PBS with 10% FBS as previously described 7 . Typically, AGM from pooled embryos of 1-2 litters (8-20 AGM) is necessary to obtain su cient numbers of EC. 8. Perform uorescent activated cell sorting (FACS) by rst gating SSC and FSC (using relatively broad gates as embryonic EC tend to vary in size) and gating live cells as DAPI negative. Gate cells as positive for VE-cadherin, and negative for CD41, CD45, and Ter119 (to minimize contamination from hematopoietic populations). Use isotype control antibodies to determine thresholds for setting gates for sorting. Set the sorting machine to the lowest ow rate of 1.0 to avoid excess shear stress on the sorted EC. Sort cells into a 5mL tube containing cold PBS/10% FBS.
Plating Endothelial cells 1. 12-24 hours prior to sort add 0.25 ml Retronectin (5 micrograms/ml in PBS) to individual wells of 48well tissue culture plate. Incubate at 4°C overnight. Prior to adding cells remove retronectin solution and wash with 0.5 ml PBS. Do not let well dry. Add and remove PBS to individual wells at a time.
2. Centrifuge sorted cells at 300g for 5 minutes. Resuspend 2 x 10^5 cells/ml in AGM-EC+ media. Remove PBS from 48 well and add 0.5 ml cells in AGM-EC+ media to each well. ECs initially seem to be density sensitive and grow better if not too sparse (target at least 1 x 10^5 EC per 48 well). Generally, at least two wells of ECs should be plated, reserving one well as a control (mock infection).
Infecting Endothelial cells 1. Culture EC with AGM-EC+ media for 1-2 days in a 37°C tissue culture incubator with 5% CO2. Cells surviving the sort form scattered adherent colonies/clusters of dividing ECs. To remove dead cells, it may be necessary to remove media and add fresh AGM-EC+ media on day 1-2.
2. When cells have dispersed and cover 50-75% of the plastic bottom (sub-con uent, typically at 1-2 days), infect with PGK.myr-AKT lentivirus (described in Kobayashi et al. Nat Cell Biol, 2010; see Butler and Ra i, 2012, for lentiviral production protocol). Remove stored viral supernatant (in -80°C) and immediately place in ice to thaw. While virus thaws, remove media from wells of ECs and add 0.35 ml AGM EC+ media + 0.5 microliters protamine sulfate (4 mg/ml [4 micrograms/ml nal]). Add 0.15 ml viral supernatant to one well. Add 0.15 ml AGM-EC+ media to control wells (mock infection). Incubate 24 hours in a 37°C tissue culture incubator with 5% CO2.
3. Remove media containing virus and replace with 0.5 ml AGM EC media (base media without additives). 4. Culture for 1-2 days in a 37°C tissue culture incubator with 5% CO2 until cell layer evenly covers the bottom of the well (con uent) (see Figure 1).
Culturing AGM Akt-EC cells 1. Once infected cells in 48 well are con uent, add 1 ml 0.1% gelatin to 1 well of a 12 well plate and incubate at room temperature for 15 minutes. Aspirate gelatin solution and leave the plate in a tissue culture hood with the lid removed until the wells are dry.
2. Dissociate cells in the 48 well by adding 0.2ml TrypLE solution. Incubate in a 37°C tissue culture incubator for 3-5 min until cells are detached. Pipette to a 15 ml conical tube and add 9 ml AGM-EC media. Centrifuge cells at 300 g for 5 min, aspirate supernatant and resuspend the pellet in 2 ml AGM-EC media and plate in one gelatinized well of a 12 well plate. Culture in a 37°C tissue culture incubator with 5% CO2.
3. Once cells are con uent in 12-well, transfer cells (reserving 4X10^4 cells for serum-free viability testing in step 4 below) to 1 well of 6 well plate using trypsinization procedure described above and culture in a 6 well until con uent, then transfer to 1-T25 tissue culture ask and culture until con uent and then transfer to 1 T75 tissue culture ask.
4. The PGK.myr-AKT vector contains no uorescent marker, so mock infections are generally done as a control. PGK.myr-AKT infected cells will passage well, whereas after the rst passage from the 48 well, mock infected ECs generally stop expanding and will not survive in serum-free conditions. For this reason, after passage to a 12 well format, test whether AGM Akt EC survive and remain as a stable layer in serum free conditions required for co-culture with hematopoietic populations: plate 4X10^4 AGM Akt ECs to one gelatinized well of a 24-well plate with AGM EC media and culture in a 37°C tissue culture incubator with 5% CO2 for 24 hours. Aspirate AGM EC media from well and add X-Vivo 20 media (serum free media for culturing hematopoietic populations). Similarly, replace AGM EC media with X-Vivo 20 media in the well containing mock-infected cells. The EC monolayer in the well containing AGM Akt-EC should remain intact/con uent with X-vivo for at least 7 days, although there may be some oating/dead cells that accumulate centrally after a few days culture. In contrast, mock infected cells will die and oat away after a few days of culture with X-vivo. 5. Maintain cells in T75 tissue culture asks. Approximately every 4-7 days, once the layer is con uent, cells should be passaged. Dissociate layer with TrypLE and estimate cell number with a hemocytometer. Replate 5x10^5 cells to each gelatinized T75 ask. For co-culture experiments use AGM Akt EC cells that have been passaged fewer than 10-12 times. High passage cells tend to lose contact inhibition and grow in layers or form mounds, at which point capacity to support HSC seems to be diminished. 6. Once established, phenotype AGM Akt EC cells with ow cytometry to assess EC purity using ECspeci c antibodies including: VE-Cadherin, Flk1, and CD31, with goal of >99% purity.
2. AGM-Akt EC cells are frozen in solution containing 10% DMSO, which is cytotoxic. To prevent cell death during the thawing process, add EC media wash to cells immediately after thawing to dilute DMSO, spin down, and resuspend cells in EC media promptly.
3. During co-culture in serum-free media, there may be some dead/ oating ECs that tend to congregate centrally. This is normal as long as the EC layer remains intact and con uent during the full co-culture period.
Time Taken 8-10 days for setting up timed matings and embryo development prior to dissection and sort.
1-2 days for AGM EC culture prior to infection.

Anticipated Results
Please refer to our previous publications 1, 3 for anticipated results following co-culture of AGM-derived hemogenic precursor cells with AGM-Akt-EC (both using bulk-sorted hemogenic populations and clonal single cell co-culture by index sorting). Information on the engraftment and phenotypic properties, CFU output, and limit dilution transplants of the resultant hematopoietic progeny can be found here. AGM Akt-EC layers. Three representative images of AGM Akt-EC layers. Includes an example of a healthy, con uent AGM Akt-EC layer that is suitable for co-culture, and two examples of layers that are too overgrown to be used in co-culture experiments. These images can be used as a reference for researchers maintaining AGM Akt-EC for use in co-culture experiments.